Introduction
We have already demonstrated that an immunosupressant FK506 (tacrolimus) inhibited ischemic neuronal injury 1 , and that mild hypothermia enhanced the neuroprotective effect in the transient focal cerebral ischemia model 2 . However, mechanisms of the neuroprotective effects have not been clarified. The aim of the present study is to investigate anti-apoptotic effects of FK506 following transient focal ischemia in rats using immunohistochemical study.
Methods
A transient (2hours) focal ischemia was induced by left middle cerebral artery occlusion using an intraluminal suture method 2 . FK506 (0.3 mg/kg) or vehicle was intravenously administered immediately after occlusion. After 24 hours of reperfusion, hematoxylin and eosin staining for infarct volume measurement and immunohistochemistry for the detection of single strand DNA breaking (SSB) were carried out. To confirm characters of SSB positive cells, the double staining for SSB with anti-apoptotic protein Bcl-2, neuronal marker NeuN, or TUNEL were also performed.
Results
FK506 did not affect cortical cerebral blood flow measured by a Laser Doppler flowmetry. The total infarct volume was significantly reduced in the FK506-treated group (92.2 +/− 16.02 mm3) compared with the vehicle-treated group (136.8 +/− 13.2 mm3, p<0.01). The total numbers of SSB positive cells in the ischemic hemisphere were 135.8+/−45.7 in the FK506-treated group, and 345.3+/−43.7 in the vehicle-treated group with a statistically significant difference (p<0.01). SSB positive cells were predominantly distributed in the peri-infarct area of the ischemic hemisphere. Semi-quantified analysis revealed a significant up-regulation of Bcl-2 expression in the FK506-treated group compared with the vehicle-treated group. Cells with strong cytoplasmic Bcl-2 expression did not show nuclear SSB signals in the peri-infarct area. SSB positive cells were also stained with NeuN, and several SSB positive cells were TUNEL positive.
Conclusion
The present data demonstrated that FK506 significantly reduced infarct volume in the rat transient focal ischemia model. The neuroprotection was associated with a significant decrease in SSB positive neurones together with a significant increase in cytoplasmic Bcl-2 expression. These suggest that anti-apoptotic mechanisms are involved in the neuroprotective effects of FK506 in the model.